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Abstract

We had developed Optical Scatter Imaging (OSI) as a method which combines light scattering spectroscopy with microscopic imaging to probe local particle size in situ. Using a variable diameter iris as a Fourier spatial filter, the technique consisted of collecting images that encoded the intensity ratio of wide-to-narrow angle scatter at each pixel in the full field of view. In this paper, we replace the variable diameter Fourier filter with a digital micromirror device (DMD) to extend our assessment of morphology to the characterization of particle shape and orientation. We describe our setup in detail and demonstrate how to eliminate aberrations associated with the placement of the DMD in a conjugate Fourier plane of our microscopic imaging system. Using bacteria and polystyrene spheres, we show how this system can be used to assess particle aspect ratio even when imaged at low resolution. We also show the feasibility of detecting alterations in organelle aspect ratio in situ within living cells. This improved OSI system could be further developed to automate morphological quantification and sorting of non-spherical particles in situ.

Figures (10)

Digital micromirror device- (DMD-) based optical scattering imaging system. The DMD is placed in conjugate Fourier plane F”. Unscattered light is blocked at F’ and F” (light blue beam). The magnification of the Fourier plane on the DMD is controlled by lenses L2 and L3, while the magnification of the final image was varied by changing the focal length of L4. The imaging beam (red trace) is collimated on the DMD before final focusing onto the charged coupled device (CCD) camera.

(a) Spheres (0.465μm) imaged using a filtered halogen lamp at 632.8 ± 0.5nm (1500ms exposure). (b) The same spheres imaged using the He-Ne laser at 633nm (300ms exposure). (c) cross section of the signal in panel (a) at dashed red line, (d) cross section of the signal for the same sphere in panel (b). Each pixel corresponds to 0.27μm.

Dark field image of 0.465 μm spheres with transform occupying (a) 760 mirrors in diameter centered on the DMD, (b) 300 mirrors in diameter centered on the DMD or (c) 300 mirrors in diameter positioned at the upper right hand corner of the DMD. In (c), the DMD was translated so that the center of the shifted active aperture was aligned with the incident beam. Thus, the light reflected from the DMD’s active aperture in (c) still passed through the center of L4. In each case, insets show the position of the DMD apertures; the white area indicates ‘on’ mirrors; the black area ‘off’ mirrors.

(a) Field of view showing image overlap due to diffraction at the DMD. The green spots are the pixels chosen as point spread function during deconvolution. Note that the spots were enlarged for clarity, only one pixel at the center of each spot was actually used for the point spread function. (b) Same image as in (a) after deconvolution. (c) pixel histograms of the areas within the red and blue squares in panel (a). (d) pixel histograms of the same image areas in panel (b).

Images of the background using the He-Ne laser as light source (a) without the spinning diffuser (100ms exposure), and (b) with spinning diffuser (2000ms exposure). The background consisted of a water sample sandwiched between a microscope slide and coverslip. Lower panels show the cross section of the signal at the red dashed line in each corresponding image.

Dark field images of (a) 0.465μm sphere, and (b), (c) E. coli and high and low magnification. Corresponding intensity response, ρ, of a pixel located at the center of the object as a function of Gabor filter orientation, φ, for (d) the sphere and (e) & (f) the E. coli (e corresponds to b; f to c). The symbols connected by the solid line represent the measured data points. The dotted line indicates the expected diametrically symmetric responses at the complex conjugate frequency positions of the Gabor-like optical filters. In (e) and (f) the responses were normalized to the maximum intensity.

Images of same iBMK cells as in Fig. 9 (a) color coded image encoding aspect ratio of scattering response at each pixel. The aspect ratio was measured as maximum/average intensity of filtered responses, (b) Mitotracker Green fluorescence to visualize specifically the mitochondria. The two segmented areas include round and punctate mitochondria (Area 1) or filamentous looking mitochondria (Area 2). (c) Aspect ratio distribution for the pixels in Area 1 and Area 2. Pixel numbers were normalized to the total number of pixels within each area.